INHIBITORS OF PHOSPHATIDIC ACID PHOSPHOHYDROLASE (PAP), INCLUDING D-PROPRANOLOL AND ANALOGS THEREOF, ALONE OR IN COMBINATION WITH DESIPRAMINE, FOR THE TREATMENT OF CANCERS THAT DEPEND ON THE EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR), ONCOGENIC VARIANTS THEREOF AND OTHER MEMBERS OF THE ErbB/HER FAMILY

Abstract

Compounds and combinations of them that inhibit phosphatidic phosphohydrolase (PAP) enzymatic activity are formulated into pharmaceuticals useful in cancer treatment. Inhibitors of PAP can be used for blocking the progression of cancers that depend on the epidermal growth factor receptor (EGFR), its oncogenic variants and other members of its ErbB tyrosine kinase receptor family, through induction of their endocytosis, thus making them inaccessible to the extracellular stimuli that promote maintenance and progression of cancer.

Description

The present invention describes the use of inhibitors of fosfatidic acid phosphodydrolase (PAP) enzymes, either as single compounds or as combinations of them, for the formulation of medicines useful for cancer treatments.

The invention proposes to utilize PAP inhibitors to block the progression of cancers that depends on the epidermal growth factor receptor (EGFR), its oncogenic variants and other members of the ErbB/HER family of tyrosine kinase receptors, inducing their endocitosis and thus making them inaccessible to stimuli that promote cancer progression.

Among PAP inhibitors part of this invention are drugs currently in use for other clinical purposes, unrelated between them, except for the fact of being cationic amphiphilic molecules. These include propranolol, desipramine and chlorpromazine, each used in clinics for other medical purposes. The present invention encompasses all known PAP inhibitors, including sphingosine and bromoenol lactone.

A specific example of the present invention corresponds to D-propranolol used alone or in combination with desipramine. D-propranolol combined with its racemic mixture with L-propranolol constitutes the drug known as propranolol, a general beta-blocker that is used for treating hypertension and other pathologies. However, only L-propranolol is useful as blocker of beta-adrenergic receptors (beta-blocker), which is the activity responsible for the anti-hypertensive effects. D-propranolol contained in this invention lacks a useful beta-blocker activity.

The present invention demonstrates that a combination of D-propranolol with desipramine, results useful for inhibiting the progression and causing death of cancerous cells that depend on the EGFR or its oncogenic variants, (e.g. the EGFRvIII variant).

As such, the invention represents a second use of known drugs, used for other clinical purposes, but also includes any other kind of PAP inhibitor, such as analogs of D-propranolol that lack beta-blocker activity. Any kind of PAP inhibitor can be used alone or in combination between them or with anti-tumoral drugs having distinct mechanisms of action, including hormones, antibodies and drugs used in chemotherapy.

PAP inhibitors of present invention also can be used in combination with other kind of treatment, including surgery and radiotherapy, simultaneously of afterwards. These compounds serve not only as complement to chemotherapy or to drugs directed to block EGFR function and its oncogenic variants, but can also be the only available therapy once cancerous cells have developed resistance to drugs against the EGFR currently in use.

The invention opens new possibilities for treating cancers such as those from head and neck, colon, lung, breath, prostate, stomach, liver and glioblastomas, which frequently display with frequent oncogenic alterations of the EGFR (over-expression or mutations) or its family member ErbB2/Neu.

FIELD OF THE INVENTION

The invention encompasses the following fields:

• • 1) The biology of cancerous cells, which is determinant in the pathogenicity of cancer, frequently involving oncogenic signaling functions of EGFR and members of its family of tyrosine kinase receptors (ErbB/HER). Such alterations enhance the aggressiveness and malignancy of tumoral cells and are also crucial for their growth, survival and progression towards metastatic stages, thus offering an effective target for designing novel anti-cancer strategies, as proposed by this invention;
  • 2) Endocytic trafficking, which plays a crucial role in the normal and pathogenic functions of the EGFR and other members of its ErbB/HER1-4 family;
  • 3) The function of the PA/PKA (Phosphatidic acid/protein kinase A) signaling pathway, which has been almost unknown since the demonstration in the present invention that is involved in the regulation of EGFR accessibility to external stimuli. This novel function of the PA/PKA signaling pathway constitutes the fundament of the present invention.
  • 4) The use of PAP inhibitors for treating cancer. This has not been previously proposed.
  • 5) Second use for drugs that are already known and in use for other clinical purposes based on mechanisms of action distinct from PAP inhibition. Second application to already known and used in clinical practice for drug mechanisms of action other than inhibition of PAP. For instance, propranolol is a racemic mixture of L- and D-propranolol Propranolol where only the L-propranolol is a non-selective beta-adrenergic blocker receptors and are responsible for their effectiveness in the treatment of hypertension, arrhythmia etc. Another example is desipramine, which is commonly used as a blocking norepinephrine reuptake inhibitor and a muscarinic cholinergic receptor, as prescribed by anti-depressive treatment.

The fundament of the present invention involves all these fields interrelated with the novel function discovered for the PA/PKA signaling pathway and the novel regulation mechanisms that determine the cell surface levels of EGFR. These regulation mechanisms are susceptible of pharmacologic manipulation to the aim of inducing endocytosis and do so inaccessible EGFR activation by its external ligands.

The invention consists in the use of PAP inhibitors, such as D-propranolol and desipramine, drugs originally used for other purposes, to design novel anti-cancer therapies. This strategy has not been used previously.

Advantages of the Invention

The present invention proposes a distinct strategy from those currently in use to reduce the progression of EGFR-dependent cancers. The current drugs in use and those found in the design stage are directed directly to the EGFR molecule, attempting to block either the interaction with the ligand or the activity of its domain tyrosine kinase which is the generator of oncogenic signals. In contrast, the compounds proposed in the present invention do not have the molecule of the EGFR as direct target but instead molecules involved in the endocytic regulation of its cell surface accessibility.

This strategy fits into the present tendency of targeted treatments directed to counteract mainly the signaling pathways altered in cancerous cells. The present invention proposes to block the phosphatidic acid phosphohydrolase (PAP) activity as strategy to induce the removal of EGFR from the cell surface through endocytosis.

Another advantage is the availability of several molecules with known structure that share the capability of PAP inhibition, thus providing structural information for designing a novel family of PAP-inhibitors, by introducing an action mechanism not previously used for such purposes.

An additional and important advantage is the selectivity of selectivity of treatment for particular types of cancer; the invention is targeting a specific process involving EGFR oncogene function. This provides the opportunity to personalize the treatments according to known molecular markers, which can be analyzed in each case optimizing the use of drugs in cancers with higher probabilities of response. The application of PAP inhibitors restricted to cancers that present oncogenic alterations of the EGFR (overexpression or mutations) has clear economic advantages.

Finally, the use of compounds that act upon molecules not directly involved in carcinogenesis, might delay or avoid the development of resistance to the treatment, which is a main problem for drugs that directly target oncogenic kinases.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Propranolol and desipramine induce endocytosis of the EGFR in cultured tumoral cells. The image illustrates the immunofluorescent pattern of the EGFR in HeLa cells incubated in the absence or presence 75 μM propranolol, 75 μM desipramine, 50 μM metoprolol or 50 μM timolol.

FIG. 2: The enantiomers L-y D-propranolol as well as desipramine decrease the cell surface availability of the EGFR assessed by radioligand binding. A. L-y D-propranolol; B. desipramine; C. Combination of different concentrations of D-propranolol (upper scale) with desipramine (lower scale); D. Combination of EC₅₀ of D-propranolol and desipramine.

FIG. 3: Glioblastoma cells U87 and U87 transfected to overexpress the oncogenic mutant EGFRvIII (U87-EGFRvIII) as tumoral models with different proliferation rates.

FIG. 4: Effect of D-propranolol and desipramina added as discontinuous treatment on the viability of glioblastoma cells U87 and U87-EGFRvIII and not-tumoral epithelial cells MDCK.

FIG. 5: Continuous treatment with low concentrations of D-propranolol (Prop) and desipramine (Des), both alone and in combination, selectively inhibits the enhanced proliferation of U87-EGFRvIII and tumoral cells in primary culture from a patient with glioblastoma (GBM1) that overexpress EGFR.

FIG. 6: Continuous treatment with two concentrations (10, 30 μM) D-propranolol, close to those found in blood of patients treated for arrhythmia, used alone or in combination with 1 mM desipramine selectively inhibits the proliferation of several tumoral cell lines and tumoral cells in primary culture from a patient with glioblastoma (GBM1) that overexpress EGFR.

FIG. 7: Novel treatment strategy for EGFR-dependent cancers based on the effects of D-propranolol and desipramine.

BACKGROUND OF THE INVENTION

Cancer Problem

Cancer encompasses a group of diseases generated by uncontrolled and invasive growth of abnormal cells, which without effective treatment can result in patient's death within short periods of time (<12 months or even in 5-9 months in the most malignant cases). Thus, by frequency and mortality rate constitutes a serious problem of public health in the world.

The most frequent cancers of women are breath, cervix, colorectal and lung cancers, whereas in men are lung, prostate, stomach and colorectal cancers (1,2). As a cause of death, cancer occupies the third place after cardiovascular and infectious diseases, but taking only developed countries it is generally the second cause of death after cardiovascular diseases (1,2). Statistics for year 2002 shows 10.9 millions of new cases and 6.7 millions of deaths (3,796,000 men y 2,928,000 women), within a prevalence of 24.6 millions suffering from some kind of cancer this year (1). Among the cancers that caused most deaths were lung (1,179,000 deaths), stomach (700,000), liver (598,000) and colorectal (528,000) and among the most malignant, with less than 20% survival at 5 years, are those of lung, esophagus, stomach, liver and glioblastomas (1-3).

In Chile is not known with accuracy or incidence and survival of patients. Every year there are about 35,000 hospitalizations and 14,000 deaths from cancer. In the past 40 years, mortality rates have increased from 86 to 123 per 100,000 habitants (2000) estimated 36,500 new cases per year. (MINSAL, 2005, (4)).

Current Treatments are Usually not Effective Against Advances Cancers with High Degree of Malignancy

Treatment of cancer includes ablative surgery, radiation, chemotherapy, hormone-therapy, biologic therapies and targeted therapies, which have generated new expectations for highly malignant cancers whose response to traditional treatment is relatively poor. The fact that cancer remains an important cause of death reflects inefficient treatment, especially in advanced and highly malignant cases.

The new anticancer drugs can extend the response rates and global survival in certain cancers but still a variety of cancers maintain high levels of malignancy and mortality. Annually more than 10 million new cases are diagnosed and die around 6 million for this cause. These numbers will increase gradually in the future due to population growth, longer life expectancy (involving aging) and increased exposure to risk factors. It is estimated that the number of new cases may rise to 50% in the next 20 years, reaching 15 million by 2020. In industrialized countries, one in four people will die from cancer (5) (2).

New drugs that have been incorporated to chemotherapeutic treatments of which achieve in some cases extend overall survival of patients and response rates but still high levels of uncontrollable malignancy remain in many types of cancers. Noted for their malignancy cancers of the lung, stomach and glioblastomas, which have lower survival rates of 20% in 5 years and high mortality rate within 9 months of diagnosis. It is essential to try to improve the treatment of these cancers seeking new strategies.

Lung Cancer:

About 30% of all cancer deaths are due to lung cancer, the leading cause of cancer death worldwide (6). About 90% of lung cancers are due to smoking affecting about 1.3 billion people in the world and it annually kills about 5 million people aged 30 or older (7). In Chile, this is outweighed by the stomach cancer in men (25.3 versus 17/100.000 inhabitants) and women (13.8 versus 7.4/100,000), and biliary tract cancer in women (17.1 versus 7.4/100,000). Cancers of non-small cell lung cancer (NSCLC) is one of the advanced malignant tumors with a major risk of death. Without treatment, patients with NSCLC and metastases have a median survival of 4-5 months. Only 10% of the patients survive the year. The standard first-line therapy for advanced or metastatic NSCLC is based on chemotherapy in combination with platinum-duplex, which increases the median survival to 8-11 months and the survival rate to about 30% at a year and 14-20% at two years (8).

Gastric Cancer:

Gastric carcinoma is one of the most common epithelial-derived cancers (9). Its incidence has been declining over the past 50 years but still ranks the fourth in the frequency and the second after lung cancer as a cause of death, with significant differences between gender and ethnic groups. Each year about 1 million new cases are diagnosed and there are about 800,000 deaths from this cause. In Chile it ranks the first cause of death in males, with a rate of 25.3 per 100,000 inhabitants, and the second place in women, after cancer of the gallbladder, with a rate of 13.8 per 100,000 inhabitants (4, 10). The annual mortality rate for gastric cancer in Chile has remained stable in recent years, reaching 3,115 deaths in 2003 (11). The survival rate at 5 years for patients with localized cancer is close to 60% while for those with metastases is only 2% (12). In cases of advanced disease, without treatment, the median survival is less than 12 months (often just 5.4 months). The new chemotherapeutic modalities have failed to lower the median survival that has remained largely unchanged over the past 10-20 years. There is no established chemotherapy for this cancer (13-15).

Glioblastomas

The “gliomas” include all tumors that are presumed to have glial cell origin and they are the most common tumors of the central nervous system. The grade III (anaplastic astrocytoma) and grade IV (glioblastoma) are considered to be malignant gliomas (3). Glioblastomas have a frequency of 3/100,000 people per year and they are among the most lethal tumors of all human cancers. The median survival of glioblastoma has been maintained for decades at about 9-12 months. Only 2% of patients 65 years and over and 30% of those under 45 survive 2 years, while about 75% die 18 months after diagnosis (16,17). The invasive character and poor response to standard treatments including radiotherapy and chemotherapy contribute to the poor prognosis (18). The current treatment of radiotherapy and concomitant administration of temozolomide, followed by adjuvant temozolomide, increases the median survival of 12.1 months to only 14.6 months (19). In Chile, Lorenzoni et al (20) demonstrated that the survival of patients does not differ from those reported in other countries that have a more sophisticated diagnostic and surgical system and the best of modern technology. Therefore, the possibility of obtaining better treatment results is not dependent on diagnostic or surgical capabilities.

Cancer Treatment Using Specific Molecular Targets

The increased knowledge of cancer at the cellular and molecular level opened new treatment expectations addressing specific molecular targets and personalizing the approach according to oncogenic lesions.

Cancer cells derive from genetic damage that determines acquisition of new properties, diverging from normal behavior. In general, three kind of genes can lead to tumorigenesis: (i) oncogenes, (ii) tumoral supresor genes (anti-oncogenes); y (iii) genes involved in genetic stability (21,22). Alterations in these genes result in the acquisition of a malignant phenotype characterized by the following abnormal properties: 1) Self-sufficiency in growth signals; 2) Insensivity to anti-growth signals; 3) Evading apoptosis; 4) Limitless replicative potential; 5) Sustained angiogenesis; 6) Tissue invasion and metastasis (22).

Despite of all these abnormal properties, cancerous cells become literally “addict” to the hyperactivity of a particular network of internal signals. Fortunately, this can be used to counteract their malignancy with drugs specially designed to inhibit the corresponding hyperactive network (23-26). It is possible to obtain maximal benefits with minimal secondary effects, provided that the altered genes that sustain the hyperactive signaling network are susceptible to become identified in each tumor (27). Identification of genetic lesions is relatively advanced for some crucial signaling pathways, thus allowing to personalize the use of drugs to specific targets, either as first line or in combination with chemotherapies (28).

There is now a tendency and widespread consensus towards the needs of personalizing the cancer treatments in base of the knowledge of the crucial genetic lesions, as well as of the frequently altered molecular and biochemical processes that distinguish cancerous from normal cells (21) (22).

Protein Kinase as General Target for Directed Therapies

The main targets of directed therapies are protein-kinases, crucially involved in signal transduction systems.

The most studied molecules recognized by their importance in processes of proliferation, survival, migration and metastasis of tumoral cells are indeed enzymes with protein-kinase activity that phosphorylate serine, threonine or tyrosine residues. The genes of these enzymes and their direct regulators include oncogenes and anti-oncogenes (29). Examples include PI3KCA oncogenic molecules, EGFR and B-RAF, RAS oncogene family that activate PI3K both as Raf kinases, and the PTEN tumor suppressor which inhibits PI3K signaling (29).

Kinase inhibitors have improved the treatment of a select group of cancers, but still their efficacy against certain cancers of high malignancy is below the theoretical expectations (29). Furthermore, even the groups that benefit from treatment with these drugs resistance is frequently developed within relatively short time periods. Tumoral cells escape from the effects of kinase inhibitors due to counteracting mutations in the molecular targets that make them resistant. It is therefore required for to search for other strategies beyond direct inhibition of the oncogenic kinases.

The EGFR Kinase

The EGFR is the paradigm of tyrosine-kinases that control cellular processes critical for the development and maintenance of the malignant tumoral phenotype. EGFR is member of the receptor tyrosine-kinase family compose by HER1/EGFR/ErBB1/, HER2/Neu/ErBB2, HER3/ErBB3 and HER4/ErBB4. The EGFR traverse once the membrane and thus exhibits an extracellular domain that interact with ligands and an intracellular domain bearing the tyrosine-kinase domain (30). It is mainly localized in the plasma membrane where it is activated upon interaction with extracellular ligand stimuli. Ligand binding induces homo or heterodimers with each other and the intracellular tyrosine kinase domain is activated, starting point signaling pathways regulating proliferation and survival of cells.

The EGFR is one of the most ubiquitous receptors regulators of cell proliferation, survival and migration, all involved in tumorigenesis. EGFR is activated by at least seven distinct ligands, including EGF, HB-EGF and tumoral growth factor alpha (TGF-α) frequently secreted by tumoral cells (31). The EGFR is also trans-activated by a variety of stimuli of other receptors, specially receptors coupled to GTPases (GPCRs) (32). In our laboratory we have shown that a ubiquitous regulatory system as composed of extracellular nucleotides, including ATP and P2Y₁ receptor is able to trans-activate the EGFR (33).

Therefore, the role of EGFR should be under strict control and failure of these systems or alterations in the receptor itself may determine and carry signals exaggerated, or accompany oncogenic cell transformation that originates and sustains the cells cancer (23, 30).

These receptors are predominantly found on cell surfaces which receive and interpret processes stimuli that regulate proliferation, migration, and cell survival, all critical in the pathogenesis of cancer. Therefore, drugs capable of causing internalization from the cell surface to intracellular compartments prevent its interaction with external stimuli and change its location to oncogenic signaling. In these conditions, from the active EGFR signaling could be altered and be toxic for the tumor cell.

Alterations in the EGFR Associated to Cancer

Alterations in the function of EGFR are frequent and crucial to the pathogenesis of various solid cancers.

EGFR has been one of the most studied and most promising molecules as targets for a new generation of anti-cancer drugs because many types that have a solid cancers and oncogenic altered function of this receptor (34) (27) (35). The most frequent alterations include overexpression (36, 37) or genetic mutations resulting from re-repair or replacement of specific amino acids (38-40). When EGFR function is altered by over-expression or by activating mutations, exaggerated signals which emits intracellular, leading to the development, maintenance and invasion of tumor cells (34).

Almost 40-50% of solid tumors depend on an exacerbated activity of the EGFR tyrosine-kinase determined by genetic alterations. A detailed recount of the EGFR genetic alterations include: (i) An increased number of EGFR gene copies leading to over-expression of the receptor. Gliomas and lung tumors frequently exhibit such gene amplification (20, 21). However, other yet unknown mechanisms of EGFR over-expression exist without gene amplification, as seen in gastric cancer (36,37). However, there are other mechanisms not elucidated overexpression, as would occur in gastric cancer where EGFR gene amplification is rare and even then exhibit over-expression of this receptor associated with increased malignancy (13,14); (ii) Mutations resulting in hyperactive EGFR; a) EGFRvIII mutant that by deletion lacks the extracellular region which conform the domain of ligand binding, being most important in glioblastomes (40); b) EGFR^L858R mutant where leucine 858 is substituted by argninine and EGFR^DelE746-A750 mutant with deleted exon 19 that eliminates the conserved sequence LREA (DelE746-A750; EGFR^Del), both mutations affecting the tyrosine-kinase domain of the receptor (EGFR^TKDmut) and seen in 10-15% of non small cell lung cancer (NSCLC) (38,39).

Cells bearing genetic alterations of the EGFR become “addict” to the exaggerated (oncogenic) signals arising from the receptor. Inhibition of EGFR exaggerated activity damages more cancerous than normal cells. Therefore, inhibitors of EGFR function now constitute the paradigm for developing targeted cancer therapies (25) (41), which can be personalized after the identification of EGFR alterations in each tumor, thus optimizing the response to treatment.

Drugs Currently in Use Against the EGFR for the Treatment of Certain Cancers

The first anti-EGFR drugs developed in the 80s and approved by the FDA include two types of antagonists (35). (i) Monoclonal antibodies that inhibit ligand binding (Cetuximab y Panitumumab). Cetuximab (erbitux; Merk KGaA, Darmstad, Germany; WO2009099649), is a monoclonal humanized antibody that binds EGFR with high affinity and competitively block ligand binding. It also induces endocytosis and negative regulation of the EGFR. It is mainly used to treat advanced colorectal carcinomas that express EGFR (42,43); (ii) Small molecules that inhibit the tyrosine-kinase of EGFR, such as Erlotinib y Gefitinib (35) (WO03103676 y WO2005117887). These drugs compete with ATP for binding to the tyrosine-kinase domain. This results in the inhibition of signal transduction processes involved in proliferation, survival, and cell migration, cancer altered. Tyrosine-kinase inhibitors can be used to treat metastatic NSCLC and colon, head and neck and pancreas cancers (28,35).

Other patents of anti-tumoral drugs that inhibit EGFR function WO 03097855, U.S. Pat. No. 5,795,898.

EGFR as Target for Personalized Therapies

As EGFR is an integral part of intracellular signaling pathways that control malignant cell growth (23, 30) has become one of the most studied targets for targeted, personalized therapies (35). Two notable examples are non-small cell lung cancer (NSCLC) and colon cancer, in which the analysis of EGFR oncogenic mutations helps deciding whether tyrosine-kinase inhibitors can be conveniently used (44-46).

In NSCLC, only 10-20% of patients respond to erlotinib or gefitinib, significantly improving overall survival, progression-free survival, symptoms and quality of life (47, 48). Cetuximab is less effective (49). Responsive patients express EGFR^TKDmut receptors bearing any of the mentioned mutations in the tyrosine-kinase domain, which not only provide oncogenic properties but also sensitize the receptor to the drugs (39, 50, 51).

About 90% of the mutations of tyrosine kinase domain (EGFRT^KDmut) sensitizing treatment with erlotinib and gefitinib are the already mentioned substitution of leucine for arginine at position 858 (L858R; EGFR^L858R) or deletions in exon 19 that eliminate LREA conserved sequence (delE746-A750; EGFR^Del) (39). Apparently these mutations constitutively activate EGFR tyrosine kinase but also increase their avidity for erlotinib and gefitinib, resulting in hypersensitivity to treatment (39). These patients have response rates close to 80-90% survival of about 80% per year and a median survival that can be increased to 28 months (44, 45, 52-54). Identification of these mutants allows personalized treatments that can achieve close to 80-90% responses and improved survival.

Limitations of Available Anti-EGFR Drugs

The main limitation of current drugs that counteract the oncogenic function of EGFR are a relative low efficacy, as a great proportion does not respond, and development of resistance of initially sensitive tumors.

Evidence has shown that EGFR is a good anti-tumor target but also that there are serious limitations on the drugs currently in use, stimulating research into new and complementary strategies to interfere with their function (27, 28).

For instance, those patients suffering from NSCLC that do not have depend on EGFR mutations that sensitize to erlotinib or gefitinib, which account for almost 85-90% of the cases, there is no much to offer. This is also true for other cancers that do not respond to these kinds of inhibitors. Furthermore, it is frequent to observe that even those initially sensitive patients that respond to erlotinib or gefitinib the tumoral growth recovers within periods of 6 months to 2 years. Almost 50% of these resistant tumors display a second mutation that substitutes methionine 790 for threonine in the tyrosine kinase domain, which block the interaction of the drugs with the ATP binding site. Recent studies report new drugs that now inhibit the EGFR T790M mutant (55). However, the possibility of resistance development to the new drugs still persists, as this is a common problem for kinase inhibitors (29). On the other hand, there are yet another 50% of cases that developed resistance but without the T790M mutation, in which the mechanism remains unknown (56-58).

All this indicates that EGFR is a good target for the design of antitumor drugs but it is necessary to investigate alternative and complementary to existing strategies. Currently being tested numerous other agents that target EGFR function following the same strategy, ie, blocking ligand binding or tyrosine kinase activity of the receptor (35).

EGFR Endocytosis as Target for Anti-Tumoral Drugs

The endocytic route is preponderant among the multiple mechanisms that control EGFR function. Ligand binding induce EGFR dimerization leading to activation recycle or enter into the lysosomal degradation route, thus spending variable and controlled periods of signaling before degradation (59,60). Therefore, endocytosis provides controlled mechanisms for both attenuating and compartmentalizing EGFR signaling. Because of this important role, endocytosis offers good opportunities for pharmacological intervention upon EGFR function with the aim of designing novel anti-tumoral therapies, but so far they only cover endocytosis induced by antibodies directed directly to the EGFR (61).

We recently described a novel mechanism of control of EGFR endocytosis, which involves the signaling pathway of phosphatidic acid phosphohydrolase (PAP) enzymatic activity towards down-regulation of protein kinase A (PA/PKA pathway). This mechanism can be triggered by pharmacologic inhibition of PAP, as this enzyme hydrolyses PA to diacylglycerol. A decrease in PKA activity, is the crucial step in the induction of EGFR endocytosis by small drugs inhibitors of PAP.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes the inhibition of PAP with D-propranolol alone or combined with desipramine as a novel strategy, based on endocytic induction, to block growth of cancerous cells that depend on the EGFR or its oncogenic variants.

Propranolol (which represents the racemic mixture of D and L propranolol) has been used as beta-clocker, i.e. focusing on its active principle is L-propranolol instead of D-propranolol, for experimental attenuation of tumor growth and metastasis of cancerous cells that express beta-adrenergic receptors. In these experiments, propranolol only prevents the beta-adrenergic stimulus presumed to promote tumorigenesis mimicking stress conditions, a rather controversial notion (62,63). The international publication WO 2006/017185 describe several compounds that display anti-proliferative effects in in vitro, among which desipramine is mentioned. However, this publication does not refer to any mechanism by which desipramine might be causing a decrease in cell proliferation. The international publication WO 2008/112297 describe inhibitors of the enzyme acid sphingomielinase among which appears desipramine as analogous compound, predicting that it might have anti-cancer effects, but no specific results are reported.

The inventors of the present invention have published that propranolol as racemic mixtures or L- and D-propranolol induces endocytosis of EGFR, showing that the mechanism involves PAP inhibition, an increment of PA and a subsequent increase in the activity of type 4 phosphodiesterases (PDE4), leading to a decreased cAMP levels and a decreased PKA activity (64). This novel control system gives the possibility to use different enzymes of the PA/PKA pathway, including PAP, as novel targets to induce EGFR endocytosis and decrease the growth of tumors whose malignancy depends on oncogenic EGFR function.

The invention is also based on the following additional observations: 1) The enantiomer D-propranolol, which lacks the beta-blocker activity of L-propranolol, and desipramine, both used as inhibitors of PAP, induce EGFR endocytosis; 2) D-propranolol and desipramine inhibit the proliferation of cancerous cells expressing oncogenic alterations of EGFR, including EGFR over-expression and the EGFRvIII mutant. The effect of these drugs is selective to malignant cells, leaving non-tumoral cells relatively unaffected or weakly affected.

D-propranolol and analogs lacking beta-blocker activity, alone or in combination with desipramine, could be used to complement treatments based on chemotherapies or EGFR inhibitors and also as single therapy when resistance to current treatments has already develop.

The invention opens new possibilities for the treatment of cancers whose malignancy frequently depend on oncogenic alterations of EGFR or ErbB2, including cancers of colon, lung, breast, prostate, stomach, head and neck, liver and glioblastomas.

The inventors of the present invention had previously demonstrated that PKA inhibition induces internalization of EGFR (65). More recently, they demonstrated that increases in phosphatidic acid achieved by inhibiting PAP enzymatic activity, which hydrolyses PA to diacylglycerol, reproduces the effect of PKA inhibition leading also to EGFR internalization in the absence of ligand. The mechanism involves a PA-mediated activation of type 4 phosphodiesterases (PDE4) leading to decreased cAMP levels and PKA activity (64). This PA/PDE4/cAMP/PKA signaling pathway has been previously described but its function had remained unknown (66). The investigator's results indicate that this PA/PDE4/cAMP/PKA signaling pathway regulates the cell surface levels of EGFR acting upon the receptor endocytosis and recycling (64).

Propranolol and desipramine used as PAP inhibitors (66), active the PA/PDE4/cAMP/PKA signaling pathway that induces EGFR endocytosis (64). This use of propranolol and desipramine is completely distinct from their currently use in clinical treatments. For instance, propranolol is clinically used to treat hypertension because of the beta-blocker effect of L-propranolol. Desipramine is used to block recapture of noradrenaline in depression treatment. Inhibitory effects on PAP enzymatic activity have been describes for Propranolol and desipramine, including chlorpromazine and esphingosine (66,68). However, propranolol and desipramine at high concentrations (10-70 μM), far over the blood concentrations (1-2 μM) found in patients under hypertension or depression treatments. Propranolol is currently used as PAP inhibitor to study the function of PA and diacylglycerol in a variety of cellular processes, such as signaling, protein trafficking and cytoskeletal functions (69).

Among the variety of signaling pathways where PA participate, the activation of PDE4 by PA leads to decreased levels of cAMP and consequently to decreased activity of PKA, effects that are evoked by propranolol, independently of its beta-blocker effect, and by desipramine, independently of its effect upon noradrenaline recapture (66,67).

EXAMPLES

Example 1

Propranolol and Desipramine Used as PAP Inhibitors Induce Endocytosis of EGFR

Indirect immunofluorescence of FIG. 1 shows that propranolol (L and D racemic mixture) induce a redistribution of EGFR from the cell surface to an intracellular predominantly perinuclear location, which has been defined as recycling endosomes by its co-localization with transferring, a marker for these kind of endosomes (64). This effect is not due to the beta-blocking activity because it is not reproduced by other beta-blockers such as methoprolol y timolol. Desipramine that also has an inhibitory effect upon PAP activity (66, 67) mimics the endocytic effect of propranolol.

So far, all known PAP inhibitors provoke the same effect as propranolol and desipramine, thus extending the range of the invention to any PAP inhibitor. The results shown here are only an example, which does not restrict or limit the field of the invention.

It has recently been reported that the racemic mixture of propranolol (L/D) causes a decrease in binding of the radioligand ¹²⁵I-EGF for 30 min which becomes 80% at the highest dose (64).

Decreased radioligand binding activity induced by propranolol shows one of the fundamentals of the invention, thus making EGFR inaccessible to extracellular ligands and consequently reducing its oncogenic role.

Example 2

Comparative Assessment of L-, D-Propranolol and Desipramine Effects on EGFR Endocytosis Measured by Radioligand Binding

The effect of propranolol as inhibitor of PAP, proposed by the present invention for blocking the tumorigenic action of EGFR, requires higher concentrations than those as reported as beta-adrenergic blocker in vitro (62,63). However, the results of the inventors demonstrate L-propranolol and D-propranolol are equivalent in their capacity of reducing the cell surface of EGFR (FIG. 2, A), both with an EC₅₀ of about 75-100 μM.

The beta-blocking effects of propranolol (racemic L and D mixture) used in clinics relays only on the effectiveness of L-propranolol (70). D-propranolol is also a reported PAP inhibitor (67), but can be used at higher doses.

Desipramine is also effective inducing a decrease in radioligand binding, with EC₅₀ of about 20 μM (FIG. 2B). A combination of EC₅₀ for desipramine (20 μM) and propranolol (75 μM) decreases 75-80% of EGFR from the cell surface, as assessed by ¹²⁵I-EGF binding assay (FIGS. 2C y D).

Therefore, combinations of D-propranolol and desipramine can be more effective than each drug alone for treating cancers that depend on the oncogenic stimulus of EGFR or its oncogenic variants through endocytosis.

Example 3

Tumor Cell Models with Different Proliferation Rates Dependent on EGFR and its Oncogenic Variant EGFRvIII

The cell line from human glioblastoma, U87, expresses low levels of EGFR (data not shown) and when transfected to overexpress the mutant EGFRvIII the rate of proliferation increases enormously (FIG. 3). These cells provide an appropriate study model to test the drugs.

The other model system used was from glioblastoma multiform cells, derived directly from a patient (GBM1), that express high level of EGFR but not EGFRvIII (data shown).

Example 4

Antitumoral Effect of D-Propranolol and Desipramine in Discontinuous Treatments in Glioblastoma Cells in Culture

The effectiveness of D-Propranolol and Desipramine as inhibitors of proliferation and viability of tumor cells dependent on the EGFR was demonstrated in glioblastoma cells in culture.

D-Propranolol (60 μM) alone or combined with Desipramine (2 μM) administered to cell culture for 60 min twice daily inhibits both proliferation and viability of the U87 cells as it does to the U87-EGFRvIII cells (FIG. 4).

The sensitivity to the combination of D-Propranolol and Desipramine is greater in the U87-EGFRvIII cells. As a control of selectivity, the experiment shows that MDCK cells, a canine kidney cell line that expresses low levels of EGFR and that are not malignant (33), are not affected by the drugs alone and their viability decreased 20% with the combined treatment. These results indicate selectivity of the treatment in glioblastoma cells, which is increased by expression of the oncogenic variant EGFRvIII.

Example 5

Antitumoral Effect of D-Propranolol and Desipramine in Continuous Treatments and Low Doses in Glioblastoma Cells in Culture

To inhibit the proliferation and cellular viability, significantly lower concentrations can also be used in more prolonged treatments. Concentrations as low as 2 μM Propranolol (only double of what is described in the plasma of patients under treatment for hypertension) together with 0.6 μM Desipramine (half of that described in plasma) achieve remarkable effects in U87-EGFRvIII cells, without greatly affecting the cells that do not express this oncogenic mutant (FIG. 5).

This surprising effect of the combination of both drugs makes it possible to use clinically accepted doses for cancers that express the EGFRvIII.

In summary, the D-Propranolol (and analogs that lack beta-blocker activity) and Desipramine, alone or in combination are useful for the treatment of cancers whose malignancy depends on oncogenic alterations of the EGFR, given either by over-expression (such as BMB1) or by mutations (e.g., EGFRvIII).

The scope of the invention extends to cancers that express other oncogenic EGFR variants, such as those described in lung cancer, or even other family members (e.g., ErbB2/Neu), and other inhibitors of PAP (Sphingosine and Chlorpromazine).

DETAILED DESCRIPTION OF FIGURES

FIG. 1: Propranolol and Desipramine induce endocytosis of the EGFR in tumor cells in culture: The immunofluorescence image shows the distribution of the EGFR in HeLa control cells and cells treated for 30 minutes with the indicated drugs. Both Propranolol (75 μM) and Desipramine (75 μM) induce a change in distribution of the receptor from the cell surface (Control), indicated by staining of the cell borders, into intracellular compartments, indicated by vesicular and perinuclear staining. Metoprolol (50 μM) and Timolol (50 μM), other beta-adrenergic blockers, have no effect.

FIG. 2: The enantiomers L- and D-Propranolol and Desipramine reduce the availability of EGFR at the cell surface determined by radio ligand binding: HeLa cells previously deprived of serum for 4 hours were treated for 30 min with the indicated doses of the different drugs and then a radio ligand binding assay was performed incubating the cells with 20 ng/ml ¹²⁵I-EGF at 4° C. for 1 hour, to detect the EGFR at the cell surface. A. L- and D-Propranolol caused a drop in the binding of ¹²⁵I-EGF reflecting a decrease of about 50% of EGFR at the cell surface at a concentration of 75 μM (EC₅₀) and 80% at 250 μM. The effects of L- and D-Propranolol are indistinguishable; B. Desipramine also induced a drop in the levels of radio ligand binding, with EC₅₀˜20-25 μM; C. Combination of different concentrations of D-Propranolol (upper scale) and Desipramine (lower scale); D. Combination of D-Propranolol (75 μM) and Desipramine (25 μM) used in their respective EC₅₀ caused a ˜75% decrease in the binding of ¹²⁵I-EGF.

FIG. 3: Glioblastoma cells U87 and U87 transfected to overexpress the oncogenic mutant EGFRvIII (U87-EGFRvIII) as a model of tumor cells with different proliferation rates. 10,000 cells were seeded in 24-well plates with MEM medium supplemented with 10% FBS and antibiotics (Penicillin 100 U/ml and Streptomycin 100 mg/ml). The U87 cells permanently transfected with plasmid pcDNA3-EGFRvIII-myc (U87-EGFRvIII) show higher proliferation than U87 cells.

FIG. 4: Effect of D-propranolol and desipramine in discontinuous treatment upon viability of tumoral glioblastoma U87, U87-EGFRvIII and normal epithelial MDCK cells.

5000 cells (U87, U87-EGFRvIII y MDCK) were seeded in 96-well plates in MEM medium supplemented with 10% FBS and antibiotics (Penicillin 100 U/ml and Streptomycin 100 mg/ml). Treatment with 60 μM D-Propranolol (Prop), 2 μM desipramina (Des) or a combination of both was made for 1 hour twice a day over three days. Cell viability estimated with a crystal violet colorimetric assay is expressed as % of untreated cells (Control). Each drug decreases viability about 60% of U87 an U87-EGFRvIII cells but not MDCK cells. The combination of both drugs is notably more effective against U87-EGFRvIII cells, killing more than 95% of them, while MDCK cells decrease by only 20%.

FIG. 5: The continuous treatment with low concentrations of D-Propranolol and Desipramine selectively inhibits the exaggerated proliferation of U87-EGFRvIII and glioblastoma GBM1 cells that overexpress the EGFR. After seeding 2,000 cells (U87, U87-EGFRvIII, MDCK and GBM1) per well and cultured under the same conditions as FIG. 3 for 24 hours, the culture medium was replaced without (control) or with the indicated drugs, 2 μM D-Propranolol (Prop) or 0.6 μM Desipramine (Des) or the combination of both, changing the medium every 24 hours for 6 days. The viability was estimated using crystal violet assay and is represented as % compared to untreated controls. The combination of D-Propranolol and Desipramine decreased the viability by almost 5 times for U87-EGFRvIII cells and by half for GBM1 cells, while U87 cells only decreased by 10% and the MDCK were not affected.

FIG. 6: New strategy for treatment of EGFR-dependent cancers based on the effects of D-Propranolol and Desipramine: In contrast to current strategies that are directly targeting the EGFR molecule, attempting to inhibit ligand binding or tyrosine kinase activity, the invention proposes to induce the endocytosis of the receptor mediated by drugs that inhibit PAP and activate the PA/PKA signaling pathway, thereby inducing a relocation of EGFR from the cell surface to endosomes.

DESCRIPTION OF TECHNIQUES

Reagents and Antibodies

Recombinant human EGF was purchased from Invitrogen (Carlsbad, Calif.), protein A-Sepharose, propranolol and high glucose DMEM from Sigma-Aldrich (St. Louis, Mo.), fetal bovine serum from Hyclone Laboratories (Logan, Utah), cell culture reagents from Invitrogen and Sigma Aldrich, plastic plates for tissue culture from Nalge Nunc (Naperville, Ill.). The culture medium of hybridoma HB8506 purchased from the American Type Culture Collection (ATCC, Manassas, Va.) was used as a source of monoclonal antibodies against the extracellular domain of human EGFR. The rabbit monoclonal antibody against the carboxyl terminal domain of EGFR was purchased from Millipore.

Construction of Plasmid for the Expression of EGFRvIII Oncogene

EGFR cDNA cloned into the pBK-CMV vector between HindIII and SmaI sites described in (56) was used for constructing the expression vector for oncogenic variant EGFRvIII. First, PCR fragments corresponding to exons 1-2 and 7-28 were obtained using primers containing restriction sites for KpnI and EcoRV for the first fragment and EcoRV and XbaI for the second fragment. After ligating these fragments a new fragment was obtained corresponding to EGFRvIII that lacks exons 2-7 and then was cloned between KpnI and XbaI sites in pcDNA3.1 and pcDNA3.1 myc vectors, obtaining the vectors pcDNA3-EGFRvIII and pcDNA3-EGFRvIII-myc, respectively. Both constructs were analyzed by sequencing.

Cell Culture and Transfection

HeLa cells which we used for the radioligand binding assays and EGFR endocytosis have been characterized previously in our laboratory (33, 64, 65). The cells were grown to approximately 80% confluence and maintained without serum for 2 h before testing. U87 human hybridoma cells purchased from the ATCC were grown and transfected with pcDNA3.1-EGFRvIII-myc. Transfection was performed with the lipofectamine method following the manufacturer's instructions (Invitrogen). 100,000 U87 cells were seeded in 15 mm plated and after 48 hours of seeding cells were transfected with 1 μg of plasmid pcDNA3-EGFRvIII-myc. After 48 hours post-transfection the selection medium (DMEM, 10% Fetal Bovine Serum (FBS) supplemented with antibiotics (100 IU/ml penicillin, 100 mg/ml streptomycin) and 0.8 mg/ml Geneticin was added G418, Sigma). After 2 weeks in selection media, permanently transfected cells were obtained (U87-EGFRvIII) and analyzed by indirect immunofluorescence using a rabbit monoclonal antibody (Millipore) against the cytosolic domain of EGFR.

GMB1 cells were obtained from a biopsy specimen from a patient diagnosed with glioblastoma multiforme. The biopsy specimen of the tumor was approximately 1 cm. It was washed 4 times with PBS, cut in pieces and incubated in PBS with 0.125% trypsin, 5 mM EDTA for 15 min at 37° C. with continuous agitation. After mechanical disruption with a Pasteur pipette, cells were centrifuged at 1200 rpm for 10 min in a tabletop centrifuge. The pellet was resuspended 2 ml of PBS with ovomucoid inhibitor (1 mg/ml) and DNase I (1 mg/ml) and then re-centrifuged again for 10 min at 1200 rpm. The cells were resuspended in NH4Cl/Tris pH 7.2 and incubated for 5 min at 37° C. (5 ml to 50 million cells), pelleted by centrifugation for 10 min at 1200 rpm, and finally resuspended and cultured in DMEM with 10% FBS. These cells were maintained for at least 10 passages.

Evaluation of Proliferation and Viability of Cultured Tumor Cells In Vitro and Treatment with D-Propranolol and Desipramine

U87 cells, U87-EGFRvIII, MDCK, and GMB1 cells were seeded at a density of 2000-10000 cells per well (as indicated in the corresponding figures) in 96-well plates and cultured in DMEM supplemented with 10% FBS and antibiotics (penicillin 100 U/ml and streptomycin 100 mg/ml. The medium was changed at 24 hours and treatments were done in a continuous or discontinuous mode. Discontinuous treatment was done by incubating the cells with concentrations of 60 μM D-Propranolol (Prop) or 2 μM desipramine (Des) separately, or a combination of 60 μM D-Prop and 2 μM Des for 1 h twice a day for three days. Continuous treatment consisted of incubating the cells with 2-30 μM D-Prop, 0.6-1 μM Des or a combination of both drugs, changing the medium every 24 h. The respective controls received only culture medium. Cell viability was estimated either by a crystal violet colorimetric assay at an optical density of 550 nm, which intensity is proportional to the number of cells adhered to the plate, or directly counting the cells in an automatic counter.

Indirect Immunofluorescence

HeLa cells grown on glass coverslips were washed three times with cold phosphate buffer saline (PBS) and incubated 2 h in serum free DMEM-HEPES media at 37° C., in order to accumulate EGFR on the cell surface. Then the cells were washed with PBS and fixed for 30 min at room temperature with 4% paraformaldehyde in PBS supplemented with 0.1 mM CaCl2 and 1 mM MgCl 2 (PBS-CM). After washing three times with PBS plus 0.2% gelatin (300 Bloom, Sigma Aldrich, PBS-CM-G), 5 minutes each time, cells were permeabilized with 0.2% Triton X-100 for 10 minutes at room temperature and incubated for 1 h at room temperature with anti-EGFR monoclonal antibody (mAb) HB8506 (1/100 in PBS-CM-G). After at least 6 washes in PBS-CM-G cells were incubated with secondary antibody anti-mouse IgG coupled to Alexa488 (dilution 1/1000) for 1 h at room temperature. Fluorescence digital images were obtained on a Zeiss Axiophot microscope with an oil immersion objective Plan-Apochromat 63X/1.4 and Zeiss Axiocam camera and transferred to 14 bits at a computer workstation running imaging software AxioVision (Zeiss, Thorn-wood, NY) as described (64).

Ligand-Binding Assays and Endocytic Rate Constants

The radioligand 125 I-human EGF was prepared by the chloramine T method as described (71), yielding specific activities of 50000-70000 cpm/ng. Binding assays were performed in Hanks solution with 20 mM HEPES and 0.1% bovine serum albumin (BSA) for 2 h at 4° C., as described (65). 4H serum deprived HeLa cells were treated for 30 min with the indicated doses of various drugs and then incubated with 20 ng/ml 125I-EGF at 4° C. in binding medium (MEM-Hank's, 25 mM HEPES, 0.2% BSA RIA grade), with vigorous stirring for 1 h. The cells were lysed with 1 N NaOH for 2 h at room temperature and the counts per minute of each sample in triplicate was determined in a gamma counter, including a nonspecific binding point obtained by incubation with an excess (500 fold) of cold ligand, whose value was subtracted from each sample. The radioligand binding in saturating condition provides an estimate of the amount of EGFR on the cell surface.

Summary of the Invention of a Novel Strategy for Cancer Treatment

Epidermal growth factor receptor (EGFR) belongs to the tyrosine kinase receptor family ErbB/HER that comprise four members (ErbB1-4 or HER1-4). EGFR is activated by at least six different specific ligands broadcasting intracellular signals that depending on cellular context can promote processes or cell proliferation, differentiation, survival, migration and apoptosis. All these processes become altered during cancerigenesis and are frequently associated to oncogenic dysfunctions determined by EGFR over-expression or hyperactive mutations. Because of this, EGFR became a preponderant target for developing new drugs and strategies for directed and personalized anti-tumoral therapies. (25) (41). Tumoral cells are literally “addicted” to oncogenic signals emitted from such altered EGFR, as reflected in their higher sensitivity to EGFR inhibitors than normal cells. Treatments can be personalized and optimized by tumor analysis to detect oncogenic alterations of EGFR. So far, the strategies has been focused on directing drugs to the EGFR molecule itself, attempting to inhibit either ligand binding with antibodies or the receptor tyrosine-kinase activity with small molecules. Drugs in clinical use include humanized monoclonal antibodies to block ligand binding (e.g. Cetuximab) and drugs that compete for ATP binding to the active site of the EGFR tyrosine kinase (Gefitinib and Erlotinib), inhibiting its activity. Although clinical data support the notion that EGFR is a good target for directed and personalized anti-tumoral treatments, the efficacy of these kind of drugs remains limited, being usually restricted to small subgroups of sensitive patients (35). An addition, a general problem to kinase inhibitors is the relatively frequent apparition of mutations conferring resistance to the treatment. Therefore, it is necessary to have novel pharmaceutics with different and complementary mechanisms of actions from those currently in use.

Our invention proposes an innovative strategy, which is to use small drugs for pharmacological inducement of EGFR removal from the cell surface through endocytic internalization, thus decreasing its accessibility to extracellular activating stimuli and changing the cellular location of signal broadcasting of already active receptors. Both alternatives can be deleterious for tumoral cells that base their malignancy on an exaggerated EGFR signaling activity at the cell surface. Drugs that induce EGFR endocytosis can inhibit proliferation and viability of EGFR-dependent tumoral malignant cells. This strategy (depicted in FIG. 5) is novel. It has not been used before, as it is based on experimental data obtained in our laboratory.

Endocytosis plays an essential role in the function of EGFR. When EGFR binds to its ligands on the cell surface is first activated and then endocytosed, remaining active for varying times while recycling the cell surface, to be finally assigned to a degradation route terminating its signaling (59). We have demonstrated that propranolol (racemic mixtures of L and D propranolol) used as inhibitor of phosphatidic acid phosphohydrolase (PAP) activity induce endocytosis and intracellular accumulation of EGFR, independently of ligand (64). We also showed that this effect depends on a signaling pathway involving increments of phosphatidic acid (PA) that activate type 4 phosphodiesterases, leading to decreased cAMP and consequently to decreased PKA activity. We have later shown that EGFR endocytosis can be also triggered by D-propranolol and by desipramine. After activation of the PA/PKA signaling pathway by PAP inhibition, the EGFR is internalized and thus becomes inaccessible to external mitogenic stimuli. These observations open the possibility of using this PA/PKA signaling pathway to design new pharmaceutical formulations to counteract the malignancy of a large proportion of cancers that depend on oncogenic alterations of the EGFR, including overexpression.

The feasibility of this strategy is demonstrated by our additional results. We have observed that D-propranol (that lacks beta-blocker activity) and desipramine, both used as PAP inhibitors, not only induce EGFR endocytosis but also inhibit proliferation and decrease viability of tumoral cancer cells that overexpress the EGFR (glioblastoma cells GBM1 derived from a patient) or the oncogenic variant EGFRvIII (U87 cells transfected for EGFRvIII). The combination of D-propranolol and desipramine results more effective.

The invention is extensive to cancers that express other oncogenic variants of the EGFR or other members of the ErbB/HER family (e.g. ErbB2/Neu). It is also extensible to other compounds that have inhibitory actions upon PAP, either already known compounds (e.g. esphingosine y chlorpromazine) or any other new compound that might appear in the future. PAP inhibitors as proposed in this invention can be used to treat a variety of cancers, including cancer of head and neck, lung, stomach, breast, liver, prostate, liver, ovary and glioblastomas. The treatment proposed by the invention can complement other treatments or be unique when resistance to chemotherapies or EGFR inhibitory drugs has already developed.

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Claims

1-12. (canceled)

13. A method of treating cancer to a subject in need of treatment for the cancer, comprising administering an effective amount of an inhibitor of the enzyme phosphatidic acid phosphohydrolase (PAP) to the subject in need of treatment for the cancer,

wherein the cancer is dependent on the epidermal growth factor receptor (EGFR), its oncogenic variants (EGFR mutants) or other ErbB/HER family members

and wherein the inhibitor is selected from the group consisting of D (+) propranolol, a combination of D (+) propranolol with desipramine, and a combination of a racemic mixture of propranolol with desipramine.

14. The method according to claim 13, wherein the combination of the racemic mixture of propranolol with desipramine is administered.

15. The method according to claim 13, wherein the combination of D (+) propranolol with desipramine is administered.

16. The method according to claim 13, wherein the cancer is selected from the group consisting of lung, breast, colorectal, head and neck, pancreas, ovarian, stomach and esophagus, hepatic, prostate, melanoma and glioblastoma cancers.

17. The method according to claim 13, further comprising administering additional drugs that inhibit EGFR, its oncogenic variants (EGFR mutants) or other ErbB/HER family members, through a pathway different from inhibition of PAP.

18. The method according to claim 13, further comprising administering a non-pharmaceutical procedure to the subject.

19. The method according to claim 18, wherein the procedure is selected from the group consisting of radiotherapy and surgery.